During development and differentiation, genes become competent to be expressed or are stably silenced in an epigenetically heritable manner. This selective activation/repression of genes leads to the differentiation of tissue types. Our group is interested in the molecular mechanisms that lead to the heritable transmission of the silenced state. To address this problem, we are studying the mechanism of gene silencing by the Polycomb group genes (PcG) in Drosophila. The PcG genes encode a diverse group of proteins known to be important for silencing of homeotic genes during development in organisms as diverse as Drosophila and man. Many PcG genes encode chromatin-associated proteins that are thought to silence transcription through modification of histones and formation of inactive chromatin. PcG proteins act through cis-acting DNA sequences called Polycomb group Response Elements (PREs). PREs contain binding sites for multiple proteins that act together to recruit PcG protein complexes to the DNA. Our lab is working to identify all the DNA binding-proteins necessary to recruit PcG protein complexes. Interestingly, PREs also mediate interactions between distant DNA elements suggesting they may bring together distant silencers and enhancers with promoters. Our lab is investigating this possibility at the Drosophila engrailed gene. Many of the PcG proteins are associated in two protein complexes that repress gene expression by modifying chromatin. Both of these protein complexes specifically associate with PREs in vivo, however, it is not known how they are recruited or held at the PRE. PREs are complex elements, made up of binding sites for many proteins. Our laboratory has been working to define all the sequences and DNA binding proteins required for the activity of a 181-bp PRE from the Drosophila engrailed gene. At least 8 binding sites are present within this 181-bp PRE. Two of the binding sites are for the Polycomb-group proteins Pleiohomeotic (Pho) and Pleiohomeotic-like (Phol). The proteins GAGA factor and Pipsqueak bind to another two sites, but the identity of the proteins that bind to the other four sites was not known. We have recently found that one of the sites necessary for PRE activity, Site 2, can be bound by members of the Sp1/KLF family of zinc-finger proteins. This family of proteins encodes transcription factors and has been extensively studied in mammals. There are 20 Sp1/KLF family members in mammals. In Drosophila there are 10 Sp1/KLF family members, and nine of them bind to Site 2. We derived a consensus-binding site for the Sp1/KLF Drosophila family members and show that this consensus sequence is present in most of the molecularly characterized PREs. These data suggest that one or more Sp1/KLF family members play a role in PRE function in Drosophila. Our laboratory is currently studying these factors to elucidate their role in PRE function. Regulatory sequences in eukaryotic genes can be located many tens of kilobases away from the promoter. How do these distant regulatory sequences activate or repress the promoter? One model is that proteins bound to a distant enhancer or silencer interact with proteins bound near the promoter causing a loop of the intervening DNA. One of the fragments of DNA from the Drosophila engrailed gene that has PRE activity can also mediate interactions between distant DNA fragments. We are currently trying to understand the function of this DNA in vivo by deleting it from the endogenous engrailed gene. Our results suggest that this fragment of DNA acts to facilitate interactions between a distant enhancer involved in positively regulating engrailed expression in the wing and the engrailed promoter. Thus, this fragment of DNA acts as a PRE (a negative regulatory element) in some transgenes, but as a positive regulatory element in its natural context. These results are consistent with the idea that the role of this element is to facilitate the interactions between enhancers or silencers with distant promoter elements.